The present invention relates to a method for detecting or monitoring the activity of an enzyme. In particular, the invention relates to the use of a polypeptide multimer capable of generating a multimerisation-dependent signal and whose multimerisation properties are modulated by the activity of a protease in such a method.
Proteolysis has long been recognised as an important intra- and extracellular modification of proteins. Endopeptidase enzymes recognise particular primary sequence signals (and sometimes also secondary or tertiary structural cues) within a substrate protein and cleave the peptide bond following a particular amino acid. Exopeptidases, on the other hand, digest polypeptides from the N or C terminus. Exopeptidases are generally not sequence-specific. These enzymes play a role in, for example, digestion, the coagulation of blood, the complement cascade and the destruction of inactive, mutated or foreign forms of proteins in the cell. Proteolysis is also important as a method of recycling amino acids within the cell for the synthesis of new proteins or for utilisation as a fuel source. More recently, the role of proteolysis in signalling and in specific intracellular processes has been recognised.
It is clear that aberrant proteolysis plays a significant role in a number of disease processes. Examples include the processing of xcex2-amyloid precursor protein (inappropriate processing of this protein is thought to play a role in Alzheimer""s Disease), the inappropriate activation of proteolytic enzymes of digestion leading to pancreatitis and a loss of proteolysis of the insulin receptor precursor leading to diabetes. Proteolysis is now understood to play important roles both within the cell and in processes important in homeostasis in multi-cellular organisms. These include:
Production of bioactive molecules from inactive precursors. A hallmark of proteolytic enzymes is their production in many cases as inactive proenzymes and their subsequent rapid activation by a proteolytic event. This may be an autocatalytic process or part of a cascade. This is exemplified by the blood clotting cascade and also the cleavage of digestive proproteases to their active form. One of the central events in acute pancreatitis is the premature proteolysis and activation of pancreatic enzymes (especially trypsin) leading to autodigestion of pancreatic tissue amongst other effects (Acute pancreatitis, Mergener, K. and Baillie, J. British Medical Journal (1998) 316 44-48). Proteases are also known to activate other proenzymes and to play a role in the generation of other bioactive molecules. An important clinical example of this is the generation of angiotensin II by the enzyme angiotensin converting enzyme (ACE). ACE cleaves the C-terminal two residues from the inactive angiotensin I to produce the active form, angiotensin II. Angiotensin II has potent vasoconstrictive and salt-retentive properties, the control of ACE activity by ACE inhibitors has an important clinical role in the treatment of hypertension, heart failure, myocardial infarction and diabetic nephropathy (Angiotensin converting enzyme inhibitors, Brown N J. and Vaughan, D E., Circulation (1998) 97 1411-1420).
Destruction of bioactive molecules. An important aspect of a regulatory process is the presence not only of an xe2x80x98on switchxe2x80x99 but also the potential to switch it off again. This is an area in which proteolysis is particularly important as it is an irreversible modification. The only way in which the process can be restarted is by a resynthesis of the destroyed component. This affords a high level of control over timing. An important clinical example of this is the degradation of bradykinin by ACE. Bradykinin has a number of effects in the body including inducing smooth muscle contraction, increasing vascular permeability and promoting vasodilation and natriuresis. This, together with the example above, indicates that ACE is important in the regulation of the balance between the antagonistic effects of angiotensin II and bradykinin (Angiotensin converting enzyme inhibitors, Brown N J. and Vaughan, D E., Circulation (1998) 97 1411-1420).
Protein turnover. The ability of the cell to degrade unwanted, damaged or foreign proteins is of great importance in the maintenance of the cell. Limited proteolysis of foreign proteins is also important in the antigen presentation process and therefore in an appropriate immune response to pathogens.
Post-translational modification. The proteolysis of certain proteins is key in their ability to perform their function in the cell. For example, the biosynthesis of the insulin receptor involves the cleavage of a large precursor to produce the subunits of the receptor complex (Biosynthesis and glycosylation of the insulin receptor, Hedo, J. A., Kahn, C. R., Hayashi, M., Yamada, K. M., Kasuga, M., Journal of Biological Chemistry (1983) 258 10020-10026). The assembly of the plant lectin concanavalin A (con A) also involves the proteolysis of a precursor protein and the religation of fragments in an altered order to generate the mature protein (Traffic and assembly of concanavalin A, Bowles, D. J. and Pappin, D. J., Trends in Biochemical Science (1988) 13 60-64).
A process coincident with other forms of post-translational modification. Proteolysis is an important feature of the processes leading to the addition of glycosylphosphatidylinositol (GPI) anchors to proteins and also in some fatty acylation reactions (such as farnesylation or geranylgeranylation).
Thus, proteolysis is an important post-translational modification of proteins and peptides which occurs both within and outside of the cell and can be an essential part of other forms of post-translational modification such as addition of a GPI anchor or some fatty acids. The ability to measure the cleavage of a protein or peptide at a specific site where that protein or peptide is also accessible for the addition of a prenyl moiety or a GPI anchor will allow the in vitro and in vivo study of processes for which the methods currently available are limited.
However, methods presently available for monitoring or detecting protease activity are not sufficiently sophisticated to be useful. Reporters are currently available to follow proteolysis where a peptide containing the cut site of the protease of interest has fluorophores at either end. Modification is followed by a change in the fluorescent output on cleavage of the peptide (causing physical separation of the fluorophores). Methods are available for monitoring both in vivo and in vitro proteolysis given the availability of various chemical fluorophores and quenchers and also a number of GFP variants which can be expressed in the cell (Compositions for the detection of proteases in biological samples and methods of use thereof, Komoriya, A. and Packard, B. S., WO96/13607; Tandem fluorescent protein constructs, Tsien, R. Y., Heim, R. and Cubitt, A., WO97/28261). All such methods, however, rely on the use of a synthetic reporter which is typically not the natural substrate for the enzyme being assayed. Moreover, the flexibility of the prior art systems is limited.
According to a first aspect of the present invention, there is provided a polypeptide multimer comprising a first polypeptide and a second polypeptide, wherein
a) at least one of the polypeptides is susceptible to protease digestion;
b) association of the polypeptides to form a multimer is detectable via a signal; and
c) digestion of at least one polypeptide results in modulation of the association state of the multimer and modulation of the signal.
The invention accordingly provides a polypeptide multimer, or a constituent polypeptide thereof, which is susceptible to protease digestion such that digestion leads to dissociation of the constituent polypeptides, or a part thereof, from the multimer. The dissociation and association of the polypeptides in the multimer is in turn detectable, for example via a label (further described below), or by monitoring of molecular weight, such as by surface plasmon resonance, or by measuring the molecular interactions of polypeptides through changes in emission or absorbance spectra of constituent parts thereof. For example, the association of the multimer is detectable through the interaction of labels placed on two or more polypeptides, which differs depending on whether the polypeptides are multimerised or not. For example, where the labels are fluorescent labels, fluorescence resonance energy transfer (FRET) is observable when the labels are in close proximity in a multimer. FRET is absent or otherwise modulated when the multimer dissociates.
xe2x80x9cModulation of the signalxe2x80x9d refers to the capacity to either increase or decease a measurable signal by at least 10%, 15%, 20%, 25%, 50%, 100% or more; such increase or decrease is contingent on proteolytic cleavage of at least one polypeptide component of a multimer.
The multimer may be a homomultimer or a heteromultimer. In the former, the polypeptide monomers are substantially identical, whilst in the latter they differ. Preferably, the multimer is a heteromultimer. In the context of the present invention, a xe2x80x9cmultimerxe2x80x9d may be a dimer, consisting of two monomers; however, it may optionally be a trimer, tetramer, pentamer or hexamer, composed of groups of three or more monomers, or dimers, trimers, etc. of constituent components which are themselves composed of individual monomer components. In all of these situations, the invention requires merely that the molecule (referred to as a xe2x80x9cmultimerxe2x80x9d) should be capable of moving between an associated and a dissociated state in response to digestion of a component thereof by a protease enzyme.
According to the invention, binding of a first polypeptide to a second polypeptide is dependent upon protease digestion, which digestion may occur on one or more polypeptides.
As referred to herein, a polypeptide is xe2x80x9csusceptible to digestion by a proteasexe2x80x9d if it is available for cleavage by one or more protease enzymes in accordance with the present invention. Advantageously, the polypeptide is susceptible to digestion by a specific protease enzyme, and preferably only susceptible to digestion by a specific protease enzyme. This facilitates the reduction of non-specific or background proteolysis and the use of the invention to assay specific proteolytic events.
Advantageously, digestion preferably occurs at a protease cleavable site, which may be engineered into the one or more of the polypeptide(s)xe2x80x94an xe2x80x9cengineered sitexe2x80x9dxe2x80x94or may be naturally present in one or more of the polypeptide(s)xe2x80x94a xe2x80x9cnatural sitexe2x80x9d. However, it is also possible to design one or more polypeptide(s) such that they are potentially exposed to digestion by a protease which initially recognises a site which may be distal to the polypeptide itselfxe2x80x94such as on a further polypeptide bound to a polypeptide according to the inventionxe2x80x94and/or by an exoprotease enzyme which digests non-site specifically from the N or C terminus of the polypeptide.
The term xe2x80x9cprotease cleavable sitexe2x80x9d refers to an amino acid sequence which is recognised by (i.e., a recognition site for) a protease enzyme. It is contemplated that a site comprises a small number of amino acids, typically from 2 to 10, less often up to 30 amino acids, and further that a site comprises fewer than the total number of amino acids present in the polypeptide.
An engineered protease cleavable site suitable for digestion may be placed within a polypeptide of the invention at a position such that formation of a multimer between the isolated polypeptide and its binding partner is dependent upon the intactness of the site,
It is contemplated that the position at which an engineered site is to reside may initially be determined by random placement of the site within the polypeptide, followed by testing by methods described herein of the ability of the polypeptide to associate into a multimer with its intended binding partner(s), or not, depending upon the intactness or otherwise of the site. A pair of polypeptides, of which at least one comprises a site so placed that association of the polypeptides is dependent on cleavage at this site, is useful in the present invention.
As used herein, the term xe2x80x9cpolypeptidexe2x80x9d refers to a polymer in which the monomers are amino acids and are joined together through peptide or disulphide bonds. xe2x80x9cPolypeptidexe2x80x9d refers to a full-length naturally-occurring amino acid chain or a fragment thereof, such as a selected region of the polypeptide that is of interest in a binding interaction, or a synthetic amino acid chain, or a combination thereof. xe2x80x9cFragment thereofxe2x80x9d thus refers to an amino acid sequence that is a portion of a full-length polypeptide, between about 8 and about 500 amino acids in length, preferably about 8 to about 300, more preferably about 8 to about 200 amino acids, and even more preferably about 10 to about 50 or 100 amino acids in length. Additionally, amino acids other than naturally-occurring amino acids, for example xcex2-alanine, phenyl glycine and homoarginine, may be included. Commonly-encountered amino acids which are not gene-encoded may also be used in the present invention. All of the amino acids used in the present invention may be either the D- or L-optical isomer. The D-isomers are preferred for use in a specific context, further described below. In addition, other peptidomimetics are also useful, e.g. in linker sequences of polypeptides of the present invention (see Spatola, 1983, in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Weinstein, ed., Marcel Dekker, New York, p. 267).
xe2x80x9cNaturally-occurringxe2x80x9d as used herein, as applied to a polypeptide or polynucleotide, refers to the fact that the polypeptide or polynucleotide can be found in nature. One such example is a polypeptide or polynucleotide sequence that is present in an organism (including a virus) that can be isolated from a source in nature. Once the polypeptide is engineered as described herein it is no longer naturally occurring but is derived from a naturally occurring polypeptide.
xe2x80x9cPolynucleotidexe2x80x9d refers to a polymeric form of nucleotides of 2 up to 1,000 bases in length, or even more, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA. The term is synonymous with xe2x80x9coligonucleotidexe2x80x9d.
As used herein, the term xe2x80x9cassociatesxe2x80x9d or xe2x80x9cbindsxe2x80x9d refers to polypeptides as described herein having a binding constant sufficiently strong to allow detection of binding by a detection means, such as FRET. Preferably, the polypeptides, when associated or bound, are in physical contact with each other and have a dissociation constant (Kd) of about 10 xcexcM or lower. The contact region may include all or parts of the two molecules. Therefore, the terms xe2x80x9csubstantially dissociatedxe2x80x9d and xe2x80x9cdissociatedxe2x80x9d or xe2x80x9csubstantially unboundxe2x80x9d or xe2x80x9cunboundxe2x80x9d refer to the absence or loss of contact between such regions, such that the binding constant is reduced by an amount which produces a discernible change in a signal compared to the bound state, including a total absence or loss of contact, such that the proteins are completely separated, as well as a partial absence or loss of contact, so that the body of the proteins are no longer in close proximity to each other but may still be tethered together or otherwise loosely attached, and thus have a dissociation constant greater than 10 xcexcM (Kd). In many cases, the Kd will be in the mM range. The terms xe2x80x9ccomplexxe2x80x9d and, particularly, xe2x80x9cdimerxe2x80x9d, xe2x80x9ctrimerxe2x80x9d, xe2x80x9ctetramerxe2x80x9d, xe2x80x9cmultimerxe2x80x9d and xe2x80x9coligomerxe2x80x9d, as used herein, refer to the polypeptides, peptides, proteins, domains or subunits in the associated or bound state. More than one molecule of each of the two or more polypeptides may be present in a complex, dimer, multimer or oligomer according to the methods of the invention.
As used herein the term xe2x80x9cmodulation of the association statexe2x80x9d refers to the ability of a protease enzyme, as defined above, to promote, prevent or reverse the association of at least two polypeptides, as defined above, by at least 10%, preferably by 25-50%, highly preferably by 75-90% and, most preferably, by 95-100% relative to the association observed in the absence of digestion by a protease enzyme under the same experimental conditions.
According to the experimental conditions, a monomer or multimer may change its association state by partially associating or dissociating without being either entirely reduced to the constituent monomer polypeptides or in a present exclusively in a single multimeric form. For example, a single polypeptides may dissociate from a trimer, leaving a polypeptide dimer. Moreover, cleavage of one of the monomers may occur such that the label is removed, and the signal generated on multimer formation thus modulated, but the monomer otherwise remains part of the multimer.
Alternatively, the association of polypeptides to form a multimer is inhibited by the presence of a modification which can itself be removed by proteolysis, thus allowing association of the polypeptides into the multimer. Preferably, the modification is of a residue of a coiled coil which is susceptible to cleavage from one of the polypeptides, such that the coiled coil is no longer able to form; the modification may, for example, be a phosphorylation of one of the residues. Cleavage of the coil such that the modified residue is removed leaves remaining coiled coils available for association.
The xe2x80x9cdetectable signalxe2x80x9d referred to herein may be any detectable manifestation attributable to the presence of a label and will depend on the means selected for label detection. For example, in the event that the label is detected by FRET, a label will be present on at least two polypeptide components of the multimer such that association and dissociation thereof can be monitored by measurement of energy transfer between the labels. However, if the label is detected for example by fluorescence correlation spectroscopy (FCS), which relies on the measurement of the rate of diffusion of a label, only a single labelled polypeptide is required. In the case of FCS detection, the labelled polypeptide is advantageously very much smaller than the associated multimer. For example, the labelled polypeptide is preferably between 25 and 50% of the molecular weight of the multimer, advantageously 10 to 25%, and more preferably 1 to 10% or less.
The xe2x80x9clabelxe2x80x9d according to the invention preferably comprises a light emitting detection means, and the light emitting detection means advantageously emits light of at least a fluorescent wavelength emission. It is preferred that the light emitting detection means comprises two different fluorophores or fluorescent tags or groups.
A xe2x80x9cfluorescent tagxe2x80x9d or xe2x80x9cfluorescent groupxe2x80x9d refers to either a fluorophore or a fluorescent protein or fluorescent fragment thereof. xe2x80x9cFluorescent proteinxe2x80x9d refers to any protein which fluoresces when excited with appropriate electromagnetic radiation. This includes proteins whose amino acid sequences are either natural or engineered.
It is additionally preferred that the fluorophores comprise fluorescein and tetramethylrhodamine or another suitable pair. In another preferred embodiment, the label comprises two different fluorescent proteins. It is preferred that fluorescent proteins comprise any protein selected from the group consisting of green fluorescent protein (GFP), blue fluorescent protein, red fluorescent protein and other engineered forms of GFP.
Preferably, the polypeptide comprises a cysteine or lysine amino acid through which the label is attached via a covalent bond.
Preferably, the measuring is performed by fluorescent resonance energy transfer (FRET), fluorescence anisotropy or fluorescence correlation spectroscopy.
It is preferred that the fluorescence emitting means comprise two different fluorophores, and particularly preferred that the fluorophores comprise fluorescein and tetramethylrhodamine or another suitable pair.
As used herein with regard to fluorescent labels for use in FRET, the term xe2x80x9cappropriate combinationxe2x80x9d refers to a choice of reporter labels such that the emission wavelength spectrum of one (the xe2x80x9cdonorxe2x80x9d moiety) is within the excitation wavelength spectrum of the other (the xe2x80x9cacceptorxe2x80x9d moiety).
The invention also encompasses a pair of polypeptides which associate to form a multimer, the pair comprising a first polypeptide comprising at least one binding domain, at least one site susceptible to proteolytic digestion, and a label, whereby the proteolytic digestion of at least one polypeptide is detectable via binding of the binding domain with a second polypeptide; and a second polypeptide which is capable of binding to the first polypeptide, wherein multimer formation is detectable via the label.
The invention additionally provides a method of screening for a candidate modulator of enzymatic activity of a protease, the method comprising mixing in an appropriate buffer an appropriate amount of a polypeptide susceptible to protease digestion, wherein the polypeptide binds to at least a second polypeptide, and wherein at least one polypeptide is suitably labelled with detection means for monitoring association/dissociation between the polypeptides; and a sample of material whose enzymatic activity is to be tested; and monitoring the digestion of the polypeptide.
Modulation of the association of the polypeptides to form a multimer is indicative of a modulation in the activity of the protease enzyme, and therefore of the activity of the candidate protease enzyme modulator.
As used herein, the term xe2x80x9csamplexe2x80x9d refers to a collection of inorganic, organic or biochemical molecules which is either found in nature (e.g., in a biological- or other specimen) or in an artificially-constructed grouping, such as agents which might be found and/or mixed in a laboratory. Such a sample may be either heterogeneous or homogeneous.
As used herein, the interchangeable terms xe2x80x9cbiological specimenxe2x80x9d and xe2x80x9cbiological samplexe2x80x9d refer to a whole organism or a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). xe2x80x9cBiological samplexe2x80x9d further refers to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof. Lastly, xe2x80x9cbiological samplexe2x80x9d refers to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as proteins or nucleic acid molecules.
As used herein, the term xe2x80x9corganismxe2x80x9d refers to all cellular life-forms, such as prokaryotes and eukaryotes, as well as non-cellular, nucleic acid-containing entities, such as bacteriophage and viruses.
It is highly preferred that a method of the methods described above comprises real-time observation of association of an isolated polypeptide and its binding partner or of an isolated pair of polypeptides.
As used herein in reference to monitoring, measurements or observations in assays of the invention, the term xe2x80x9creal-timexe2x80x9d refers to that which is performed contemporaneously with the monitored, measured or observed events and which yields a result of the monitoring, measurement or observation to one who performs it simultaneously, or effectively so, with the occurrence of a monitored, measured or observed event. Thus, a xe2x80x9creal timexe2x80x9d assay or measurement contains not only the measured and quantitated result, such as fluorescence, but expresses this in real time, that is, in hours, minutes, seconds, milliseconds, nanoseconds, picoseconds, etc. Shorter times exceed the instrumentation capability; further, resolution is also limited by the folding and binding kinetics of polypeptides.
A variant of the present invention as described above involves the use of first and second polypeptide binding domains, which are located on the same polypeptide, in a method according to the invention. The polypeptide is configured such that binding between the domains results in spatial rearrangement of labels present on the polypeptide, such that a signal is induced or modulated.
In a further aspect, the present invention provides a method for monitoring the activity of a protease enzyme, comprising the steps of:
a) providing a first binding domain having associated therewith a label, and a second binding domain, wherein
i) at least one of the binding domains is susceptible to protease digestion; and
ii) the first and second binding domains are capable of binding to each other such that a detectable signal is generated by the label, and digestion of one or both of the polypeptides by the protease enzyme results in modulation of the binding of the polypeptides to each other and therefore of the detectable signal;
b) allowing the binding domains to bind to each other and induce a detectable signal;
c) contacting the binding domains with a protease enzyme; and
d) detecting modulation of the detectable signal as a result of the modulation of the binding of the binding domains.
Where more than one polypeptide is used, the polypeptides are capable of associating to form a multimer. Preferably the multimer is a dimer, trimer, or tetramer. Advantageously, it is a dimer. Preferably, the label is present on two or more polypeptide constituents of the multimer.
Where the binding domains are present on a single polypeptide, it is not necessary for a multimer to be formed.
Detection of the signal attributable to the label in the binding domains may be carried out according to the invention as set forth above.
A xe2x80x9cbinding domainxe2x80x9d, as used herein, is a polypeptide domain capable of mediating the binding of one polypeptide to a second polypeptide. Exemplary binding domains are described below, and include bZIP domains, coiled coil domains, SH2 domains and SH3 domains.
As used herein, the term xe2x80x9ccontactingxe2x80x9d refers to the act of placing two reagents in such a relationship that they may potentially interact in order to produce a chemical or biological effect. Preferably, the process of contacting involves admixing the reagents at an appropriate concentration in solution or suspension, either in liquid or solid phases, or both, in an appropriate buffer.
As used herein, the term xe2x80x9cappropriate bufferxe2x80x9d refers to a medium which permits activity of the protease enzyme used in an assay of the invention, such as a low-ionic-strength buffer or other biocompatible solution (e.g., water, containing one or more of physiological salt, such as simple saline, and/or a weak buffer, such as Tris or phosphate, or others as described hereinbelow), a cell culture medium, of which many are known in the art, or a whole or fractionated cell lysate; provided that it is compatible with the binding of the components of the assay of the invention, and with the selected signal employed. For example, the buffer advantageously does not include agents which quench fluorescence, if the signal is a fluorescent signal. An xe2x80x9cappropriate bufferxe2x80x9d permits digestion of polypeptides according to the invention and, preferably, inhibits degradation and maintains biological activity or the reaction components. Inhibitors of degradation, such as nuclease inhibitors (e.g., DEPC) are well known in the art. Lastly, an appropriate buffer may comprise a stabilising substance such as glycerol, sucrose or polyethylene glycol.
As used herein, the term xe2x80x9cappropriate concentrationxe2x80x9d refers to an amount of reagent (for example, a labelled polypeptide of the invention) which is sufficient for the intended reaction to proceed in a detectable manner. For instance, in the case of a labelled polypeptide, an appropriate concentration may be considered to be that concentration at which the label emits a signal within the detection limits of a measuring device used in an assay of the invention. Such an amount is great enough to permit detection of a signal, yet small enough that a change in signal emission is detectable (e.g., such that a signal is below the upper limit of the device).
Moreover, the invention relates to a method for detecting or monitoring the activity of a modulator of a protease enzyme, comprising the steps of:
a) providing a first binding domain, and a second binding domain, wherein
i) at least one of the binding domains is susceptible to protease digestion; and
ii) the first and second binding domains are capable of binding to each other such that a detectable signal is generated by the label, and digestion of one or both of the polypeptides by the protease enzyme results in modulation of the binding of the binding domains to each other and therefore of the detectable signal;
b) allowing the binding domains to bind to each other and induce a detectable signal;
c) contacting the binding domains with a protease enzyme;
d) detecting modulation of the detectable signal as a result of the modulation of the binding of the binding domains to determine a reference signal modulation;
e) contacting the binding domains with a protease enzyme and a candidate modulator of the protease enzyme; and
f) detecting modulation of the detectable signal as a result of the modulation of the binding of the binding domains, and comparing the modulation detected with the reference signal modulation.
A xe2x80x9creference signal modulationxe2x80x9d is the amount by which a detectable signal is modulated, as defined above, in response to the activity of a protease enzyme in accordance with the invention. For example, therefore, the signal may be modulated, that is increased or decreased, by 10%, 15%, 20%, 25%, 50%, 100% or more. The reference signal modulation may be calculated at any time, and used as a standard value; it need not be recalculated every time the assay is performed. Comparison of detected signal modulation values with the reference signal modulation preferably manifest themselves as increases or decreases in the percentage modulation with respect to the reference value.
The assay permits the assessment of the activity of compounds, whether naturally-occurring or synthesised, to modulate the activity of a protease enzyme. It thus permits the use of the invention to detect or monitor processes which rely on protease activity or result in or from protease activity, such as post-translational modifications of proteins. The invention preferably relates to a method for detecting or monitoring fatty acylation of a protein, wherein the reaction includes a proteolytic event comprising the foregoing steps.
The term xe2x80x9cmodulatorxe2x80x9d thus refers to a chemical compound (naturally occurring or synthesised), such as a biological macromolecule (e.g., nucleic acid, protein, non-peptide, or organic molecule), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues, or even an inorganic element or molecule. Modulators are evaluated for potential activity as inhibitors or activators (directly or indirectly) of a biological process or processes (e.g., agonist, partial antagonist, partial agonist, antagonist, antineoplastic agents, cytotoxic agents, inhibitors of neoplastic transformation or cell proliferation, cell proliferation-promoting agents, and the like) by inclusion in screening assays described herein. The activities (or activity) of a modulator may be known, unknown or partially-known. Such modulators can be screened using the methods described herein.
The term xe2x80x9ccandidate modulatorxe2x80x9d refers to a compound to be tested by one or more screening method(s) of the invention as a putative modulator. Usually, various predetermined concentrations are used for screening such as 0.01 xcexcM, 0.1 xcexcM, 1.0 xcexcM, and 10.0 xcexcM, as described more fully below. Test compound controls can include the measurement of a signal in the absence of the test compound or comparison to a compound known to modulate the target.
xe2x80x9cModulationxe2x80x9d refers to the capacity to either increase or decease the proteolytic activity of a protease enzyme by at least 10%, 15%, 20%, 25%, 50%, 100% or more; such increase or decrease may be contingent on the occurrence of a specific event, such as activation of a signal transduction pathway, and/or may be manifest only in particular cell types.
In a still further aspect, the invention provides the use of a polypeptide multimer according to the preceding aspects of the invention for the detection or monitoring of a protease activity.
Other features and advantages of the invention are found in the detailed description of the invention, and in the following claims.